This application claims the benefit of Korean Patent Application No. 2003-76216, filed on Oct. 30, 2003, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein in its entirety by reference.
1. Field of the Invention
The present invention relates to a micro PCR device, a method of amplifying a nucleic acid using the same, and a method of measuring the concentration of a PCR product by measuring an electrical signal.
2. Description of the Related Art
Conventional PCR showing only qualitative result of amplified DNA via an electrophoresis at the end point of the reaction had many problems in terms of accuracy in the quantitative detection of DNA and the like. To resolve these problems, a real-time PCR device capable of quantitatively analyzing DNA by detecting the intensity of fluorescence proportional to the concentration of amplified DNA via an optical detecting system was developed.
Quantitative analysis of DNA is essential to the treatment of diseases and the study of DNA expression. For example, in the case of a hepatitis patient infected with the hepatitis B virus (HBV), the tolerance of the virus to an administrated drug must be regularly examined by quantitatively measuring the concentration of HBV in the patient's blood plasma via real-time RT-PCR for successful drug treatment.
Conventional real-time PCR required various optical devices such as a filter in addition to a laser source, a micromirror, and a microscope and used expensive fluorescent dyes. Many PCR chips for DNA quantitative analysis were also developed. However, conventional real-time PCR chips were based on a fluorescence detection principle, and thus had many drawbacks in terms of miniaturization and cost.
To resolve these problems, efforts to electrically detect DNA using capillary electrophoresis were made [Christa L. Colyer et al., Journal of Chromatography A, Volume 781, Issues 1-2, 26 Sep. 1997, pp. 271-276; F. Laugere et al., Sensors and Actuators B: Chemical, Volume 83, Issues 1-3, 15 Mar. 2002, pp. 104-108; Pumera et al., Anal. Chem., 2002, 74(9), pp. 1968-1971]. The electrical detection of DNA makes qualitative analysis possible but has many problems in terms of quantitative analysis. Since it is troublesome to transfer a PCR product to a capillary electrophoresis detection system via a microchannel after completing the PCR and since a high voltage is required, it costs much and miniaturization of the detection apparatus is difficult to achieve.
Milles et al. filed a patent application based on a concept that as the concentration of DNA increases during a PCR, resistance decreases and conductivity increases [U.S. Patent Publication No. 2002/0072054 A1]. It is noted that a PCR chip used in the present application is an end-point detection PCR chip rather than the real-time PCR chip. Further, an ionically-labeled probe must be used to detect a PCR product.
Meanwhile, a reproducible signal could not be detected by the conventional detection methods using an electrical or chemical signal. This is believed to be because components in a PCR mixture, such as protein, ion, and stabilizer, adsorb to an electrode surface or an inner surface of an amplification chamber. Such adsorption is very thermodynamically instable and is also affected by fluid mechanical factors. Thus, it is known that the degree of adsorption is changed, even by surrounding minor stimuli such as temperature and electromagnetic force.
The inventors found a method capable of reproducibly measuring a PCR product using an electrical signal, even at a high temperature (a maximum temperature of about 100° C.), by coating a polycationic polymer or a polyanionic polymer on the inner surface of an amplification chamber while carrying out an intensive study to resolve the problems in conventional techniques, and thus completed the present invention.